CN108165597A - The preparation method of grey mullet protein sources anti-oxidation peptide - Google Patents
The preparation method of grey mullet protein sources anti-oxidation peptide Download PDFInfo
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Abstract
The invention discloses a kind of preparation methods of grey mullet protein sources anti-oxidation peptide, include the following steps：The flesh of fish of fresh grey mullet is prepared into minced fillet, is crushed after minced fillet is lyophilized, obtains freeze-drying fish meal；Freeze-drying fish meal is subjected to degreasing using ethyl acetate, is crushed after freeze-drying, obtains degreasing grey mullet albumen powder (GM)；Degreasing grey mullet albumen powder (GM) is digested, is then centrifuged for, the freeze-drying of gained centrifugate obtains thick anti-oxidation peptide (HGM).Thick anti-oxidation peptide (HGM) is further isolated and purified, grey mullet protein sources anti-oxidation peptide can be obtained.Method using the present invention can obtain the strong grey mullet protein sources anti-oxidation peptide of antioxidant activity.
Description
Technical field
The invention belongs to technical field of functional food, are related to a kind of method that grey mullet protein sources prepare anti-oxidation peptide.
Background technology
Grey mullet (Mugil cephalus) is under the jurisdiction of spoke fin net-rope Mugiliformes Mugilidae mullet category, is pushed away by the United Nations's agriculture grain tissue
Recommend one of three big fish cultivated for world's sea water and fresh water and the Important Economic fish of east China and Southern Coast brackish culture
One of class.As one kind of low value economic fish, grey mullet is easy to cultivate, and with yield height, rich in protein and ammonia
The features such as base acid A wide selection of colours and designs.
Currently, about the research of grey mullet, number is stuck in life habit and trophic component analysis greatly absolutely, about grey mullet
The research for comprehensively utilizing and improving its added value is rarely reported, and so as to cause grey mullet utilization rate low, causes the wasting of resources.With
The progress of China's fishery cultivating technology and the development of Aquatic Product Process Industry improve the added value of low value fish to realize its synthesis
Using the inexorable trend as Fisheries Development, while opportunity also is provided for the comprehensive utilization of grey mullet, further study grey mullet money
Source, can be higher value application and the exploitation of grey mullet, and the even higher value application of other cheap aquatic products provides reason with exploitation
By foundation and reference.
In organism normal metabolic processes, body can be constantly be generated various active oxygen radicals, it is excessive from
Timely and effective removing meeting serious threat human health cannot get by base, natural is because can safely and effectively remove in human body
Excessive free radical and increasingly arouse people's interest.Thus, it has been obtained fully because safe and efficient from food anti-oxidation peptide
Development.Such as：The Chinese invention patent of patent No. ZL201210230756.6 (Authorization Notice No. CN103524596A)《It is a kind of
Shark protein anti-oxidation peptide and its preparation method and application》, (Authorization Notice No. is Patent No. ZL201310152590.5
CN103194519A Chinese invention patent)《A kind of pea protein enzymolysis prepares method and the application of anti-oxidation peptide》Etc. patents it is equal
Disclose the anti-oxidation peptide in food proteins source.However, at present there has been no oxidation resistant report is prepared by protein sources of grey mullet, with mullet
Fish prepares anti-oxidation peptide for protein sources, is of great significance for the higher value application of grey mullet with expanding anti-oxidation peptide.
Invention content
There is provided a kind of antioxidant activity strong grey mullet albumen the technical problem to be solved by the present invention is to be directed to the prior art
The preparation method of source anti-oxidation peptide.
In order to solve the above technical problem, the present invention provides a kind of preparation method of grey mullet protein sources anti-oxidation peptide, including
Following steps：
1) flesh of fish of fresh grey mullet, is prepared into minced fillet, is crushed after minced fillet is lyophilized, obtains freeze-drying fish meal；
2), degreasing：
Freeze-drying fish meal is pressed into 1g with ethyl acetate:The solid-liquid ratio mixing of 2~5ml is stirred after 50~60 DEG C (water bath condition)
100~120min is so as to carry out degreasing；It filters (remove ethyl acetate), is crushed after the freeze-drying of gained filter cake and (cross 60~100
Mesh sieves), obtain degreasing grey mullet albumen powder (GM)；
3) it, digests：
Degreasing grey mullet albumen powder (GM) and water are pressed 1:15~25 mass ratio mixing adjusts reactant as reaction system
The pH value of system is 6~8 (adjustings that pH value can be carried out with 1M NaOH), temperature is 45~55 DEG C, by 4.5~6.5U enzymes/mg degreasings
Heat preservation digests 3~5 hours after the enzyme concentration of grey mullet albumen powder (GM) adds in protease；
Enzyme deactivation after enzymolysis time reaches, is then centrifuged for, and the freeze-drying of gained centrifugate obtains thick anti-oxidation peptide (HGM).
The molecular weight overwhelming majority of the thick anti-oxidation peptide (HGM) is distributed in 10kDa hereinafter, and being accounted for less than the component of 3kDa
More than 50%.
The improvement of the preparation method of grey mullet protein sources anti-oxidation peptide as the present invention：Thick anti-oxidation peptide (HGM) is carried out
It isolates and purifies, includes the following steps：
1. by gel chromatography chromatography to grey mullet zymolyte -- thick anti-oxidation peptide (HGM) carries out initial gross separation：
HGM by glucose gel Sephadex G-25 chromatographies is detached, measures the antioxidant activity of each component,
Repeatedly chromatography collects the highest component HGM-F4 of activity；
2. HGM-F4 is further detached with reversed-phase high performance liquid chromatography, obtaining grey mullet anti-oxidation peptide, (grey mullet protein sources resist
Aoxidize peptide) HGM-F4-B4 (i.e. component B4, antioxidant activity highest).
The anti-oxidation peptide component is mainly made of the small peptide that molecular weight is 452.3434Da and 339.2600Da.
The preparation method of grey mullet protein sources anti-oxidation peptide as the present invention is further improved, 1. the step is：
It is 2.6cm × 60cm to select gel column specification, and gel media is Sephadex G-25, applied sample amount for 50~
100mg, using ultra-pure water as elution buffer, coutroi velocity 0.6mL/min collects one for every five minutes and manages, and elution time 295~
385min is component HGM-F4.
Explanation：Often pipe absorbance is detected under wavelength 280nm, each eluting peak is collected, is freeze-dried after concentration, with DPPH
Free radical scavenging activity, ultra-oxygen anion free radical clearance rate and reducing power go out the highest component of antioxidant activity for index screening.
4 components are obtained, wherein the active highest component HGM-F4 (that is, component F4) for elution time 295-385min, (see figure
5)；That is, according to test result, repeatedly loading collect highest No. 4 absorption peaks of activity (see Fig. 5) corresponding eluent.
The preparation method of grey mullet protein sources anti-oxidation peptide as the present invention is further improved：Protease is Papain
Enzyme, flavor protease, neutral proteinase, compound protease, trypsase.
The preparation method of grey mullet protein sources anti-oxidation peptide as the present invention is further improved, 2. step is：
Component HGM-F4 is detached with semi-preparative RP-HPLC, using semi-preparative column YMC-PACK ODS-AQ C18
Column, applied sample amount 5mL, elution requirement are as follows：
Wherein mobile phase A is the ultra-pure water containing 0.5% trifluoroacetic acid, and Mobile phase B contains the acetonitrile of 0.5% trifluoroacetic acid.
That is, the ultra-pure water that mobile phase A is 0.5% trifluoroacetic acid by volume content and volume content is 99.5% forms；
The acetonitrile that Mobile phase B is 0.5% trifluoroacetic acid by volume content and volume content is 99.5% forms.
The preparation method of grey mullet protein sources anti-oxidation peptide as the present invention is further improved,
In the step 3), enzyme deactivation 10~15 minutes in 95~100 DEG C of water-bath；The centrifugation for 8000~
12000r/min, 3~5 DEG C, 8~12min of centrifugation.
The preparation method of grey mullet protein sources anti-oxidation peptide as the present invention is further improved：In the step 1), newly
Fresh grey mullet, removal fish head, fish tail and internal organ, the flesh of fish are cut through high-speed homogenizer and are mixed, and obtain minced fillet；It is lyophilized in -80 DEG C to constant weight,
80 mesh sieve was crushed to, obtains freeze-drying fish meal.
Above-mentioned freeze-drying fish meal can preserve in -18 DEG C of refrigerators.
It using grey mullet meat albumen is raw material (using grey mullet as starting material) that the present invention, which is, is hydrolyzed using protease, is made
The standby anti-oxidation peptide with greater activity, including the following contents：
1) appropriate method is selected to carry out degreasing to grey mullet meat；
2) it takes appropriate grey mullet albumen powder, chooses papain, neutral proteinase, trypsase, flavor protease, compound
Protease hydrolytic grey mullet albumen using antioxidant activity as evaluation index, filters out the optimum protein enzyme for preparing anti-oxidation peptide；
3) the technique item of thick anti-oxidation peptide HGM is prepared by single factor experiment and response phase method optimization enzymolysis grey mullet albumen
Part；
4) it is detached by Sephadex-G25 and reversed-phase high performance liquid chromatography, obtains best anti-oxidant group of activity
Point.
The thick anti-oxidation peptide (HGM) that the present invention is prepared can be used as functional food base-material.Anti-oxidation peptide component HGM-F4-
B4 then has medical value.
The present invention has following technical advantage：
1), degreasing process of the invention, with common alkaline process centrifugal degreasing, alcohol degreasing, isopropanol degreasing and lipase
Degreasing method is compared, and has the advantages that degreasing rate high (84.83%), loss of proteins rate are low (14.95%).
2), thick anti-oxidation peptide HGM antioxidant activities are good, and nutritive value is high, and with preferable foaming characteristic and emulsification
Characteristic can be used as functional food ingredient.
3) it is, simple for process, it is convenient to carry out.Improve grey mullet value-added content of product.
Description of the drawings
The specific embodiment of the present invention is described in further detail below in conjunction with the accompanying drawings.
Fig. 1 is the comparison of different grey mullet degreasing process acquired results；
A, comparison of the different degreasing methods to grey mullet degreasing effect；
B, influence of the ethyl acetate degreasing time to the DPPH scavenging capacities of enzymolysis product；
C, ethyl acetate degreasing time is to the anti peroxidation of lipid ability of enzymolysis product.T0、T40、T80、T120It represents respectively de-
The fat time is the flesh of fish sample of 0min, 40min, 80min and 120min.
Fig. 2 is the activity of product of the grey mullet flesh of fish through different proteasome degradations.
Fig. 3 be under various concentration HGM-1 and HGM-2 antioxidation activity in vitro, and with GM and VCIt compares；
A, ultra-oxygen anion free radical inhibiting rate；
B, DPPH free radicals inhibiting rate；
C, hydroxyl radical free radical inhibiting rate；
D, iron ion reducing power；
E, ferrous ion sequestering power；
F, anti-lipid peroxidation ability.
Fig. 4 is the functional characteristic of HGM；
A, dissolubility；
B, foaming characteristic and foaming stability；
C, emulsibility and emulsion stability.
Fig. 5 is the separation of the thick polypeptide of grey mullet and the activity of each component；
A, Sephadex G-25 detach the thick polypeptide elution profile of grey mullet；B, the antioxidant activity of each component (F1-F4).
Fig. 6 be anti-oxidation peptide component F4 further isolate and purify and the activity of each component；
A, the reversed-phase high performance liquid chromatography figure of component F4；B, the DPPH free radical scavenging activities of each component (B1-B6)
(0.5mg/mL)。
Specific embodiment：
Below by embodiment, technical scheme of the present invention is further described in detail.
Embodiment 1：
1), pretreatment of raw material：
After fresh grey mullet clear water is cleaned, the flesh of fish is twisted into fish by removal fish head, fish tail and internal organ with high-speed homogenizer
Rotten shape, freeze-drying crush (- 80 DEG C of dryings to constant weight) and cross 80 mesh sieve afterwards, preserved in -18 DEG C of refrigerators.
2), grey mullet meat degreasing：Freeze-drying fish meal is pressed into solid-liquid ratio (1g with ethyl acetate:It 2ml) mixes, in 55 DEG C of water bath conditions
Under be stirred continuously 120min, filter cake (minced fillet after degreasing) is placed in draught cupboard after suction filtration, after ethyl acetate volatilization completely after
Frozen dried (- 80 DEG C of dryings to constant weight) is carried out, crushed 80 mesh sieve, degreasing grey mullet albumen powder (GM), -18 DEG C of refrigerator storages are made
It deposits.
3), grey mullet proteolysis：
The hydrolysising condition of GM is：Solid-liquid ratio 1:20 (mass ratioes), using neutral proteinase as hydrolase, temperature 50 C, pH
7.0th, time 5h and enzyme concentration 5.0U/mg keeps pH and temperature in hydrolytic process to stablize, the mullet obtained on this condition
Fish albumen hydrolysis solution, in 95 DEG C of water-bath enzyme deactivation 15min, 10000r/min centrifugation 10min, supernatant is freeze-dried, and (- 80 DEG C dry
It is dry to constant weight) thick anti-oxidation peptide (HGM-1).Measure the DPPH free radical scavenging activities of HGM-1 (concentration 1mg/ml) for 40.21 ±
0.36%.
The DPPH free radical scavenging activities of HGM-1, ultra-oxygen anion free radical clearance rate, the hydroxyl obtained by embodiment 1 is free
Base clearance rate, reducing power, ferrous ion chelation percent and anti peroxidation of lipid ability are as shown in Figure 3.
Embodiment 2：
The pretreatment of raw material of step 1) and the grey mullet meat degreasing of step 2) are the same as embodiment 1.
Grey mullet proteolysis：The hydrolysising condition of GM is：Solid-liquid ratio 1:20, using neutral proteinase as hydrolase, 51 DEG C of temperature,
PH7.3, time 3.5h and enzyme concentration 5.8U/mg keep pH and temperature in hydrolytic process to stablize, obtain on this condition
Grey mullet protein hydrolyzate, in 95 DEG C of water-bath enzyme deactivation 15min, 10000r/min centrifugation 10min, supernatant is slightly freeze-dried
Anti-oxidation peptide (HGM-2).The DPPH free radical scavenging activities for measuring HGM-2 (concentration 1mg/ml) are 60.19 ± 0.57%.
The DPPH free radical scavenging activities of HGM-2, ultra-oxygen anion free radical clearance rate, the hydroxyl obtained by embodiment 2 is free
Base clearance rate, reducing power, ferrous ion chelation percent and anti peroxidation of lipid ability are as shown in Figure 3.It is obtained by embodiment 2
The functional characteristic of HGM-2 is as shown in Figure 4.
Embodiment 3：
The pretreatment of raw material of step 1) and the grey mullet meat degreasing of step 2) are the same as embodiment 1.
Grey mullet proteolysis：The hydrolysising condition of GM is：Solid-liquid ratio 1:20, using neutral proteinase as hydrolase, enzymatic hydrolysis condition
For：Temperature 50 C, pH 7.0, time 4h and enzyme concentration 6.5U/mg keep pH and temperature in hydrolytic process to stablize, herein
Under the conditions of obtained grey mullet protein hydrolyzate, in 95 DEG C of water-bath enzyme deactivation 15min, 10000r/min centrifugation 10min, supernatant is through cold
Dry thick anti-oxidation peptide (HGM) is lyophilized.The DPPH free radical scavenging activities for measuring HGM (concentration 1mg/ml) are 54.57 ± 1.26%.
Embodiment 4：
The pretreatment of raw material of step 1) and the grey mullet meat degreasing of step 2) are the same as embodiment 1.
Grey mullet proteolysis：The hydrolysising condition of GM is：Solid-liquid ratio 1:20, using neutral proteinase as hydrolase, temperature 50 C,
PH7.5, time 4h and enzyme concentration 6.0U/mg keep pH and temperature in hydrolytic process to stablize, obtain on this condition
Grey mullet protein hydrolyzate, in 95 DEG C of water-bath enzyme deactivation 15min, 10000r/min centrifugation 10min, supernatant is so freeze-dried that slightly to resist
Aoxidize peptide (HGM).The DPPH free radical scavenging activities for measuring HGM (concentration 1mg/ml) are 55.43 ± 1.17%.
Embodiment 5：
The pretreatment of raw material of step 1) and the grey mullet meat degreasing of step 2) are the same as embodiment 1.
Grey mullet proteolysis：The hydrolysising condition of GM is：Solid-liquid ratio 1:20, using neutral proteinase as hydrolase, 55 DEG C of temperature,
PH7.0, time 2h and enzyme concentration 6.0U/mg keep pH and temperature in hydrolytic process to stablize, obtain on this condition
Grey mullet protein hydrolyzate, in 95 DEG C of water-bath enzyme deactivation 15min, 10000r/min centrifugation 10min, supernatant is so freeze-dried that slightly to resist
Aoxidize peptide (HGM).The DPPH free radical scavenging activities for measuring HGM (concentration 1mg/ml) are 49.74 ± 1.48%.
Embodiment 6：
2 obtained HGM-2 of embodiment is isolated and purified.
1) by gel chromatography chromatography to grey mullet zymolyte -- thick anti-oxidation peptide (HGM) carries out initial gross separation, selects solidifying
Rubber column gel column specification is φ 2.6cm × 60cm, and gel media is Sephadex G-25, and applied sample amount 100mg, elution buffer is super
Pure water, coutroi velocity 0.6mL/min collect a pipe for every five minutes, detect often pipe absorbance under wavelength 280nm, collect each elution
Peak is freeze-dried after concentration, using DPPH free radical scavenging activities, ultra-oxygen anion free radical clearance rate and reducing power as index
Filter out the highest component of antioxidant activity.4 components are obtained, wherein active highest for component F4, elution time 295-
385min (see Fig. 5).
2) component F4 is further detached with semi-preparative RP-HPLC, using semi-preparative column YMC-PACK ODS-AQ C18
Column (φ 20 × 150mm, 5 μm), applied sample amount 5mL, specific elution requirement is as shown in table 1, and wherein mobile phase A is ultra-pure water
(trifluoroacetic acid for containing 0.5%), Mobile phase B are acetonitrile (trifluoroacetic acid for containing 0.5%).Obtain 6 component B1-B6, wherein group
Divide the DPPH free radical scavenging activities highest of B4 (see Fig. 6).
Remarks explanation：
The 12nd point of 35 seconds to the 12 points eluent of 55 seconds of collection respectively, the 12nd point of 55 seconds to the 13 points eluent of 25 seconds, the
13 points of 25 seconds to 13 points eluents of 55 seconds, the 14th point of 40 seconds to the 15 points eluent of 25 seconds, the 22nd point 00 second 30 seconds to 23 points
Eluent, the 23rd point of 00 second to the 23 points eluent of 30 seconds, it is corresponding be component B1-B6.
Table 1, RP-HPLC elution requirements
Component B4 is analyzed through LC-MS it is found that it is mainly made of 2 Ala-Ile-Ala-Cys-Cys-Arg-Args 4-1 and B4-2, and molecular weight is respectively
452.3434Da and 339.2600Da
The influence of contrast experiment 1, degreasing process to grey mullet meat degreasing effect and loss of proteins rate
1), the degreasing of grey mullet meat use three kinds of method degreasings, respectively alkaline process centrifugal degreasing, organic solvent extraction degreasing and
Lipase hydrolysis degreasing, specific degreasing method are shown in Table 2.Wherein degreasing by alkali (number A, B) solid-liquid ratio is 1：4 (w/v), You Jirong
Agent extraction degreasing (number C, D, E, F) solid-liquid ratio is 1:2 (w/v), lipase (enzyme activity >=105U/g, number G) in enzyme most
Degreasing is carried out under the conditions of suitable.The corresponding degreasing flesh of fish is freeze-dried respectively after degreasing, passes through soxhlet extraction method after crushing
Fat content and protein content are surveyed with micro-Kjeldahl, calculates degreasing rate and loss of proteins rate.
Table 2, degreasing method and number
The experimental results showed that ethyl acetate degreasing method of the invention have the advantages that degreasing rate is high, loss of proteins rate it is low (see
Figure 1A).
2), influence of the ethyl acetate degreasing time to zymolyte antioxidant activity：The flesh of fish of different degreasing times is taken respectively
Dry powder 0.25g adds in 5mL phosphate buffers (pH 7.0,0.2M), keeps the temperature after five minutes, stands in 55 DEG C of constant-temperature table water-baths
The papain that mass fraction is flesh of fish dry powder 5% is added in, vibrates hydrolysis 4 hours under the conditions of 110r/min.Hydrolysis is completed
Hydrolyzate is put into enzyme deactivation 10min in 95 DEG C of water-bath immediately afterwards, rear 10000r/min centrifugations 10min is cooled to room temperature, takes
Supernatant liquor measures its DPPH scavenging capacity and anti peroxidation of lipid ability after freeze-drying.The result shows that degreasing in general
120min is preferably (Figure 1B and C).
Influence of the different protease of contrast experiment 2. to grey mullet proteolysis
It is accurate to weigh 0.5g degreasing grey mullet albumen powders with ultra-pure water by solid-liquid ratio 1:20 mixing, are put into each protease most thermophilic
5 minutes are kept the temperature in the constant temperature oscillation water-bath of degree, after being adjusted to the pH value needed for enzyme digestion reaction, a certain amount of protease is added in and starts
Hydrolysis keeps the pH value of solution constant in reaction process with 1M NaOH, and enzymolysis liquid is placed in 95 DEG C after hydrolysis 5 hours
Enzyme deactivation 15 minutes in water-bath after 10000r/min centrifuges 10min, take supernatant to be freeze-dried, freeze-dried powder is placed in -18 DEG C
It is preserved in refrigerator.
Five kinds of different protease hydrolytic grey mullet albumen are selected in this experiment altogether, and the most suitable hydrolysising condition of various enzymes is as shown in table 3,
Hydrolysing step is as described above.After the completion of hydrolysis, DPPH free radical scavenging activities (%), the water of five kinds of enzymolysis liquid dried frozen aquatic products are measured respectively
Xie Du (%), water-solubility protein concentration (mg/mL) and molecular weight distribution.
The different protease hydrolyzed action conditions of table 3
Experimental result is as shown in Figure 2.From Figure 2 it can be seen that papain has highest degree of hydrolysis, and it is 14.17%, it is neutral
The degree of hydrolysis of protease (13.47%) and compound protease (13.11%) is slightly less than papain, the hydrolysis of flavor protease
It spends minimum.Five kinds of protease zymolytes present different degrees of DPPH free radical scavenging activities, and neutral proteinase activity is most
A height of 49.92%, it is 41.69% that the activity of papain enzymolysis liquid, which is taken second place, and the activity of trypsase is minimum.And five kinds of albumen
The soluble protein concentration mensuration result of enzyme hydrolyzate is maximum for the concentration of neutral proteinase, has reached 3.3mg/mL.Comprehensive ratio
Compared with hydrolysis result is：Neutral proteinase ＞ papain ＞ compound protease ＞ trypsase ＞ flavor proteases.
Degreasing in contrast experiment 3, cancellation embodiment 1, that is, the freeze-drying fish meal obtained by step 1) is directly subjected to step 3)
Enzymolysis；Remaining is equal to embodiment 1.
The DPPH free radical scavenging activities of HGM (concentration 1mg/ml) obtained by this method are 22.7% ± 0.49%.
Experiment 1, thick anti-oxidation peptide amino acid composition analysis
The amino acid composition of HGM-2 is shown in Table 4, total amount of essential amino acids (39.99%) and essential amino acid with it is non-must
Unhydrolysed grey mullet Protein G M will be better than by needing the ratio (0.74) of amino acid.Therefore, hydrolysis not only increases grey mullet albumen
Antioxidant activity, while also increase the nutritive value of grey mullet albumen.HGM-2 is very excellent functional food base-material.
Table 4, grey mullet fish protein amino acid composition (g/100g albumen)
Note：aRepresent FAO/WHO children's proposed standard values；bRepresent FAO/WHO adult's proposed standard values；* phenylalanine is represented
The sum of with tyrosine content；∑ EAA represents essential amino acids content；∑ NEAA represents non-essential amino acid content；∑EAA/∑
NEAA represents the ratio of essential amino acid and nonessential amino acid.
Experiment 2, the antioxidation activity in vitro measure of thick anti-oxidation peptide (HGM-1 and HGM-2)
The HGM-2 of 2 gained of the HGM-1 that obtained by embodiment 1 and embodiment carries out antioxidation activity in vitro test, and with not
The GM of degradation and vitamin C (VC) make comparisons.
A, DPPH free radical scavenging activities measure：Take 2mL enzymolysis liquids (1mg/mL) and 2mL DPPH ethanol solutions
(0.1mM) is mixed, and after being positioned over light protected environment 30min, the absorbance of solution is measured under 517nm wavelength, is denoted as As.Blank group is
The absorbance of 2mL enzymolysis liquids and 2mL ethanol solutions, is denoted as A1.Control group is 2mL absolute ethyl alcohols and the anhydrous second of 2mL DPPH
The absorbance of alcoholic solution (0.1mM), is denoted as A0.The clearance rate calculation formula of DPPH free radicals is：DPPH free radical scavenging activities
(%)=[1- (AS-A1)/A0]×100。
B, superoxide anion removes experiment：Under the conditions of 25 DEG C of room temperature, the sample solution (0-2.5mg/ of various concentration is prepared
mL).Sample solution 2mL, 4.5mL Tris-Hcl buffer solutions (100mmol/L, pH8.2) and the mixing of 3mL distilled water are taken, it is to be mixed
After uniformly, the benzenetriol solution that faces that 0.5mL 3mmol/L are continuously added into solution shakes up rapidly, and measuring solution every 30 seconds exists
Light absorption value A under 325nm325, note calculate 5min internal absorbances change with time rate i.e. sample inhibition face benzenetriol autoxidation speed
Rate Vs.Sample solution is replaced with 10mmol/LHcl, the autoxidation rate for facing benzenetriol is V0.Superoxide anion clearance rate calculates
Formula is：Superoxide anion clearance rate (%)=[(V0-Vs)/V0] × 100%.
C, Hydroxyl radical-scavenging is tested：It is molten that 3.5mL distilled water, 0.5mL 9mM salicylic acids-ethyl alcohol are sequentially added into test tube
Liquid, 0.5mL sample solutions, the solution of ferrous chloride of 0.5mL9mM, 5mL8.8mM hydrogenperoxide steam generators, mixing are placed on 37 DEG C
15min is kept the temperature in thermostat water bath, the absorbance of solution is surveyed under 510nm.Scavenging action to hydroxyl free radical (%)=[1- (A1-A2)/
A3]×100.In formula：A1Absorbance for sample solution；A2The extinction that solution of ferrous chloride measures is replaced by 0.5mL distilled water
Degree；A3The absorbance that sample measures is replaced by 0.5ml distilled water.
D, reducing power measures：The sample solution of 2mL various concentrations has been added to 2mL phosphate buffers (0.2M, pH 6.6)
In the mixed solution formed with the potassium ferricyanide of 2mL1% (w/v), add 2mL10%'s (w/v) after reacting 20min at 50 DEG C
Trichloroacetic acid, mixing, 3000r/min centrifugation 10min take 2mL supernatants, add 2mL distilled water and 0.4mL mass fractions are
0.1% (w/v) iron chloride, be mixed evenly, be stored at room temperature 10min, at 700nm measure absorbance.Vc is as positive control.
E, ferrous ion sequestering power measures：It is sub- that 1mL sample solutions, the sulfuric acid of 0.05mL2mM are sequentially added in test tube
Ferrous solution, 1.85mL deionized waters add in 0.1mL5mM phenanthrene Lip river piperazine, solution are measured after placing 10min at room temperature after mixing
Absorbance A s under 562nm.With deionized water Ac as a control group, ferrous ion chelation percent (%)=[1-As/Ac] ×
100。
F, anti peroxidation of lipid ability measures：The zymolyte dried frozen aquatic products 5mg of different degreasing times is taken to be dissolved in 10mL phosphate
Buffer solution (pH 7.0,0.2M) then adds in the linoleic acid of 0.13mL and the absolute ethyl alcohol of 10mL, is mixed after being settled to 25mL with water
It is even.It places reaction liquid into 40 DEG C of biochemical cultivation case, is mixed every taking 75% ethanol solution of 0.1mL reaction solutions and 4.7mL for 24 hours
It closes, adds 0.1mL30% ammonium thiocyanates and 0.1mL 20mM solution of ferrous chloride (hydrochloric acid for being dissolved in 3.5%), mixing is equal
10min is stood after even, absorbance is measured at 500nm, BHT is as positive control.
From the figure 3, it may be seen that free radical (ultra-oxygen anion free radical, DPPH free radicals, the hydroxyl of the HGM-2 that embodiment 2 obtains
Free radical) scavenging capacity, iron ion reducing power, ferrous ion sequestering power and anti-lipid peroxidation ability it is significantly high
The HGM-1 and undegradable grey mullet albumen (GM) obtained in embodiment 1.
Experiment 3, the functional characteristic of thick anti-oxidation peptide (HGM-2)
A, solubility test：GM aqueous solutions and HGM aqueous solutions (1mg/mL) each 5mL are taken, at room temperature with 1M sodium hydroxides
Solution or 1M hydrochloric acid solutions are adjusted to pH needed for experiment (2-10), are placed 60 minutes, every 20 minutes stablizing solutions pH value with
It keeps constant.Solution is centrifuged 30 minutes in 10000g/min later, Coomassie Brilliant Blue is measured in solution and in supernatant
Protein content, the calculation formula of solubility is as follows：
B, foaming characteristic and foaming stability measure：Freeze-dried powder sample 150mg is respectively taken to be dissolved in 30mL deionized waters, at room temperature
PH to 7.0 is adjusted with 1M sodium hydroxide solutions or 1M hydrochloric acid solutions, after balancing 30 minutes at room temperature, 15000r/min dispersions
1min, after standing two minutes, measures foam volume, measures foam volume when standing 10,20 and 30 minutes respectively again later,
The calculation formula of foaming characteristic and foam stability is as follows：
C, emulsibility (EAI) is measured with emulsion stability (ESI)：The sample solution that mass fraction is 1% is prepared, with 1M hydrogen
Sodium hydroxide solution or 1M hydrochloric acid solutions are adjusted to difference pH (2-10) needed for experiment, and 30mL solution is taken to be mixed with 10mL soybean oils,
In high speed dispersor 10000r/min disperse 1min, take 50 μ L of solution from solution bottom after dispersion, immediately with 5mL mass fractions
0.1% neopelex (SDS) solution is uniformly mixed, and measures absorbance of the solution at 500nm, and make with SDS
For blank control, after solution left standstill 10min, 50 μ L solution are drawn again, measure absorbance, and emulsibility (EAI) is steady with emulsification
The computational methods of qualitative (ESI) are as follows：
ESI (min)=A0×ΔT/ΔA
Wherein：A500：Absorbance at ultraviolet 500nm；
A0：The absorbance of solution during 0min；
AT：The absorbance of solution during 10min；
ΔA：Δ A=A0-AT, Δ T=10min.
The dissolubility of protein is to evaluate the most important index of protein function characteristic, retentiveness, foaming characteristic and emulsibility
Quality both depend on dissolubility.By Fig. 4 A it is found that the polypeptide sample HGM-2 after enzymolysis be respectively provided under different pH it is higher molten
Dissolubility when Xie Xing, pH are 4 is minimum, but also reaches 89.2%.The dissolubility of GM is showed with the increase of solution ph and is first dropped
Raised trend after low, when pH value is 6, solubility is minimum, and only 15.4%.
Foaming characteristic is one of important functional character of protein, and the food such as cake, cotton candy and beer beverage are added
Work production is of great significance.As shown in Figure 4 B, largely increasing occurs in the foaming characteristic of sample after enzymolysis.Identical experiment
Under the conditions of, the foaming characteristic of degreasing grey mullet albumen powder GM is 74.1%, and the foaming characteristic of the grey mullet polypeptide HGM after enzymolysis is 121.7%.
Emulsibility is also one of important functional character of protein.Under condition of different pH, grey mullet albumen (GM) and grey mullet
The emulsibility of polypeptide (HGM-2) and the experimental result of emulsion stability are as shown in Fig. 4 Ca and Fig. 4 Cb.Under the conditions of each pH,
The emulsibility of HGM-2 is all higher than GM, but the not notable (p of difference in various degree>0.05).The emulsification of product HGM-2 after enzymolysis
Stability is significantly lower than grey mullet Protein G M (p<0.05), because peptide cannot as macro-molecular protein oil-water interfaces expansion with
Orientation, thus interfacial tension cannot be reduced, emulsion stability is caused to reduce.But in general, HGM-2 still has preferable emulsification
Stability.
Finally, it should also be noted that it is listed above be only the present invention several specific embodiments.Obviously, this hair
Bright to be not limited to above example, acceptable there are many deform.Those of ordinary skill in the art can be from present disclosure
All deformations for directly exporting or associating, are considered as protection scope of the present invention.
Claims (7)
1. the preparation method of grey mullet protein sources anti-oxidation peptide, it is characterized in that including the following steps：
1) flesh of fish of fresh grey mullet, is prepared into minced fillet, is crushed after minced fillet is lyophilized, obtains freeze-drying fish meal；
2), degreasing：
Freeze-drying fish meal is pressed into 1g with ethyl acetate:2~5ml solid-liquid ratio mixing after 50~60 DEG C stir 100~120min from
And carry out degreasing；It filters, is crushed after the freeze-drying of gained filter cake, obtain degreasing grey mullet albumen powder (GM)；
3) it, digests：
Degreasing grey mullet albumen powder (GM) and water are pressed 1:15~25 mass ratio mixing adjusts reaction system as reaction system
PH value is 6~8, temperature is 45~55 DEG C, and albumen is added in by the enzyme concentration of 4.5~6.5U enzymes/mg degreasing grey mullet albumen powders (GM)
Heat preservation enzymolysis 3~5 hours after enzyme；
Enzyme deactivation after enzymolysis time reaches, is then centrifuged for, and the freeze-drying of gained centrifugate obtains thick anti-oxidation peptide (HGM).
2. the preparation method of grey mullet protein sources anti-oxidation peptide according to claim 1, it is characterized in that：By thick anti-oxidation peptide
(HGM) it is isolated and purified, is included the following steps：
1., by gel chromatography chromatography to thick anti-oxidation peptide (HGM) carry out initial gross separation：
HGM by glucose gel Sephadex G-25 chromatographies is detached, the antioxidant activity of each component is measured, collects
The highest component HGM-F4 of activity；
2., HGM-F4 further detached with reversed-phase high performance liquid chromatography, obtain grey mullet anti-oxidation peptide HGM-F4-B4.
3. the preparation method of grey mullet protein sources anti-oxidation peptide according to claim 2, it is characterized in that, 1. the step is：
It is 2.6cm × 60cm to select gel column specification, and gel media is Sephadex G-25, and applied sample amount is 50~100mg, with
Ultra-pure water is elution buffer, and coutroi velocity 0.6mL/min collects a pipe for every five minutes, and 295~385min of elution time is
Component HGM-F4.
4. the preparation method of grey mullet protein sources anti-oxidation peptide according to claim 2, it is characterized in that, 2. the step is：
Component HGM-F4 is detached with semi-preparative RP-HPLC, using semi-preparative column YMC-PACK ODS-AQ C18 columns, on
Sample amount is 5mL, and elution requirement is as follows：
Wherein mobile phase A is the ultra-pure water containing 0.5% trifluoroacetic acid, and Mobile phase B contains the acetonitrile of 0.5% trifluoroacetic acid.
5. according to the preparation method of any grey mullet protein sources anti-oxidation peptide of Claims 1 to 4, it is characterized in that：The egg
White enzyme is papain, flavor protease, neutral proteinase, compound protease, trypsase.
6. according to the preparation method of any grey mullet protein sources anti-oxidation peptide of Claims 1 to 4, it is characterized in that：
In the step 3), enzyme deactivation 10~15 minutes in 95~100 DEG C of water-bath；The centrifugation is 8000~12000r/
Min, 3~5 DEG C, 8~12min of centrifugation.
7. according to the preparation method of any grey mullet protein sources anti-oxidation peptide of Claims 1 to 4, it is characterized in that：
In the step 1), fresh grey mullet, removal fish head, fish tail and internal organ, the flesh of fish are cut through high-speed homogenizer and are mixed, and obtain fish
It is rotten；In -80 DEG C of freeze-dryings to constant weight, 80 mesh sieve was crushed to, obtains freeze-drying fish meal.
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| CN110724178A (en) * | 2019-10-14 | 2020-01-24 | 浙江海洋大学 | Tuna white meat ACE inhibitory peptide and preparation method thereof |
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| CN113005166A (en) * | 2021-04-14 | 2021-06-22 | 中国海洋大学 | Cod polypeptide with xanthine oxidase inhibitory activity |
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